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High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

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ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Related in: MedlinePlus

Concentration response curves depicting the distinctive nuclear profiles of a cytoprotective and an apoptotic compound. (A): A compound from the Library of Pharmacologically Active Compounds screen whose nuclear profile is characteristic of a protective compound, in which nuclear parameters trend toward that of the dimethyl sulfoxide (DMSO)/vehicle control, with concentration-dependent increases in nuclear compactness and 1/(nuclear form factor), and a decreased nuclear elongation. For this compound, nuclear intensity decreases only slightly. This is not unexpected, because the difference in nuclear intensity between DMSO/vehicle-treated astrocytes and H2O2-treated astrocytes was not as significant as was seen for terfenadine treatment (supplemental online Fig. 2A). (B): Nuclear characteristics of an apoptotic compound are similar to that seen for terfenadine (supplemental online Fig. 1), with a concentration-dependent increase in nuclear intensity and elongation, and decrease in compactness and 1/(nuclear form factor). Normalized data are shown with percent activity relative to controls (0% activity = basal condition, DMSO + 12 mM H2O2-treated astrocytes).
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Figure 4: Concentration response curves depicting the distinctive nuclear profiles of a cytoprotective and an apoptotic compound. (A): A compound from the Library of Pharmacologically Active Compounds screen whose nuclear profile is characteristic of a protective compound, in which nuclear parameters trend toward that of the dimethyl sulfoxide (DMSO)/vehicle control, with concentration-dependent increases in nuclear compactness and 1/(nuclear form factor), and a decreased nuclear elongation. For this compound, nuclear intensity decreases only slightly. This is not unexpected, because the difference in nuclear intensity between DMSO/vehicle-treated astrocytes and H2O2-treated astrocytes was not as significant as was seen for terfenadine treatment (supplemental online Fig. 2A). (B): Nuclear characteristics of an apoptotic compound are similar to that seen for terfenadine (supplemental online Fig. 1), with a concentration-dependent increase in nuclear intensity and elongation, and decrease in compactness and 1/(nuclear form factor). Normalized data are shown with percent activity relative to controls (0% activity = basal condition, DMSO + 12 mM H2O2-treated astrocytes).

Mentions: We identified 42 compounds in the NPC library and 24 compounds in the LOPAC1280 library that produced protective nuclear profiles and were considered hits in the primary screen (as discussed in Materials and Methods). Eliminating duplicated compounds found in both libraries resulted in a total of 66 hit compounds (supplemental online Tables 7, 8). An example of the nuclear profile of a hit compound is shown in Figure 4A (compare with the nuclear profile of an apoptotic compound in Fig. 4B) and supplemental online Figure 9. Generally, the concentrations at which we saw maximal changes in the nuclear profile were in the high nanomolar to midmicromolar range (approximately 100 nM to 50 µM).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
Concentration response curves depicting the distinctive nuclear profiles of a cytoprotective and an apoptotic compound. (A): A compound from the Library of Pharmacologically Active Compounds screen whose nuclear profile is characteristic of a protective compound, in which nuclear parameters trend toward that of the dimethyl sulfoxide (DMSO)/vehicle control, with concentration-dependent increases in nuclear compactness and 1/(nuclear form factor), and a decreased nuclear elongation. For this compound, nuclear intensity decreases only slightly. This is not unexpected, because the difference in nuclear intensity between DMSO/vehicle-treated astrocytes and H2O2-treated astrocytes was not as significant as was seen for terfenadine treatment (supplemental online Fig. 2A). (B): Nuclear characteristics of an apoptotic compound are similar to that seen for terfenadine (supplemental online Fig. 1), with a concentration-dependent increase in nuclear intensity and elongation, and decrease in compactness and 1/(nuclear form factor). Normalized data are shown with percent activity relative to controls (0% activity = basal condition, DMSO + 12 mM H2O2-treated astrocytes).
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Related In: Results  -  Collection

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Figure 4: Concentration response curves depicting the distinctive nuclear profiles of a cytoprotective and an apoptotic compound. (A): A compound from the Library of Pharmacologically Active Compounds screen whose nuclear profile is characteristic of a protective compound, in which nuclear parameters trend toward that of the dimethyl sulfoxide (DMSO)/vehicle control, with concentration-dependent increases in nuclear compactness and 1/(nuclear form factor), and a decreased nuclear elongation. For this compound, nuclear intensity decreases only slightly. This is not unexpected, because the difference in nuclear intensity between DMSO/vehicle-treated astrocytes and H2O2-treated astrocytes was not as significant as was seen for terfenadine treatment (supplemental online Fig. 2A). (B): Nuclear characteristics of an apoptotic compound are similar to that seen for terfenadine (supplemental online Fig. 1), with a concentration-dependent increase in nuclear intensity and elongation, and decrease in compactness and 1/(nuclear form factor). Normalized data are shown with percent activity relative to controls (0% activity = basal condition, DMSO + 12 mM H2O2-treated astrocytes).
Mentions: We identified 42 compounds in the NPC library and 24 compounds in the LOPAC1280 library that produced protective nuclear profiles and were considered hits in the primary screen (as discussed in Materials and Methods). Eliminating duplicated compounds found in both libraries resulted in a total of 66 hit compounds (supplemental online Tables 7, 8). An example of the nuclear profile of a hit compound is shown in Figure 4A (compare with the nuclear profile of an apoptotic compound in Fig. 4B) and supplemental online Figure 9. Generally, the concentrations at which we saw maximal changes in the nuclear profile were in the high nanomolar to midmicromolar range (approximately 100 nM to 50 µM).

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Related in: MedlinePlus